JP2023513633A - Methods of providing personalized cells with chimeric antigen receptors (CAR) for tumor microenvironment cells - Google Patents

Abstract

The present invention is a process for providing cells containing chimeric antigen receptors (CARs) specific for one or more target antigens exposed on tumor microenvironment cells, comprising tumor microenvironment cells and non-tumor microenvironment cells. - contacting the tissue with a conjugate comprising a fluorescent moiety and an antigen-recognition moiety; By combining the steps of detecting by fluorescence emission emitted from the fluorescent moiety of the conjugate of 1 - quenching the fluorescence emitted from the fluorescent moiety of the conjugate, with antigen recognition moieties that recognize different antigens, repeating until at least two conjugates are identified that are capable of distinguishing between microenvironmental cells and non-tumor microenvironmental cells; It is directed to a process characterized by providing. Preferably, the tumor microenvironment cells are tumor stromal cells or tumor microenvironment cells derived from PaCa cells.

Description

The present invention provides cells with chimeric antigen receptors (CARs) suitable for killing/lysing tumor microenvironmental cells (TMEs) exposed on tumor microenvironmental cells (TMEs) rather than healthy cells in an individual. It relates to a method of selecting antigens that have been identified.

BACKGROUND OF THE INVENTION Cancer is a broad group of diseases involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors and invading surrounding parts of the body. Cancer can also spread to more distant parts of the body through the lymphatic system or bloodstream.

Tissues of solid tumors or cancers are usually surrounded or encapsulated by tumor microenvironment cells (TME), which consist of blood vessels, immune cells, fibroblasts, pericytes, signaling molecules and extracellular matrix (ECM). A definition of TME is disclosed in the "NCI Dictionary of Cancer Terms", National Cancer Institute, 2011-02-02. For a recent review of TME, see Am J Cancer Res. 2019; 9(2): 228-241.

Since CAR T cells are a promising tumor-targeting immunotherapy, treatment of solid tumors is affected by TME, which can, for example, act as a physical barrier or be immunosuppressive. One possible way to deal with this is to re-use CAR T cells.

The problem that arises is that tumors and TMEs are not the same for all patients, and it is necessary to select the correct patient-specific targets for both tumors and TMEs and generate CAR T cells (or similar) accordingly. That is. The selection and generation of CAR T cells must be done in a rapid manner and often patient-specifically.

In practice, single targeting to tumor cells or cells of the TME may not be sufficient, as surface molecules may also be expressed on non-target cells, raising safety concerns. In addition, surface molecules may not be selective enough, i.e., expressed on TME cells, to allow viable cells to relapse and/or cause incomplete TME destruction and/or block antigen expression levels. It is possible and/or the affinity of a single binding agent may be too low to allow efficient T cell activation.

In order to perform personalized phenotyping of tumor marker expression for individual patients, combinations of CAR and CAR T cells need to be developed within an acceptable timeframe determined by patient and indication. Clinically, the optimal duration is several weeks.

Therefore, the success of any CAR cell-based cancer immunotherapy depends on the rapid and reliable selection of specific antigens for each tumor cell.

Such a process is disclosed, for example, in EP-A-3315511, in which CAR T cells are treated with an antigen-binding region ("TCBM") according to the patient's immunological context, i.e. antigens presented by tumor cells. is provided.

A unique set of cell surface antigens have been found to be expressed on TME cells but not expressed or expressed at lower levels on non-malignant cells. These antigens (also referred to as "markers") can be used to identify and/or label and/or destroy and/or disable escape mechanisms of TME cells via ligands that specifically bind to the markers. . EP-A-3315511 does not describe the identification of the most promising antigen binding regions ("TCBMs") for therapy. Furthermore, EP-A-3315511 is directed only to tumor cells and does not describe the immunological status of TME.

After the TME cells are recognized by the CAR cells, the TME cells are thereby destroyed or at least less effective in protecting cancer cells by the CAR cells so identified.

Therefore, it is an object of the present invention to provide a method for selecting antigens specific to TME cells that are not expressed in healthy tissues in order to modify cells to kill/lyse cancer cells without attacking non-tumor cells. Met.

SUMMARY OF THE INVENTION A first subject of the present invention is a process for providing cells containing chimeric antigen receptors (CARs) specific for one or more target antigens exposed on tumor microenvironment cells, wherein the cells comprise tumor microenvironment cells. preparing a cell sample containing environmental cells and non-tumor microenvironmental cells,
- contacting the tissue with a conjugate comprising a fluorescent moiety and an antigen recognition moiety,
- removing unbound conjugate from cellular tissue and detecting cells bound to the conjugate by means of fluorescence emission emitted from the fluorescent moiety of the first conjugate;
- the step of quenching the fluorescence emitted from the fluorescent moieties of the conjugate, in combination with at least two antigen-recognition moieties that recognize different antigens, capable of distinguishing between tumor microenvironment cells and non-tumor microenvironment cells; Repeat until the conjugate is identified,
A process characterized by providing a cell having at least two identified antigen-recognizing moieties as a chimeric antigen receptor (CAR).

Preferably, the method of the invention involves repeating the above steps for at least two subsequent cycles using a conjugate comprising a fluorescent moiety and an antigen-recognizing moiety that recognizes a different antigen.

In a first embodiment, the chimeric antigen receptor (CAR) containing cells are directed to one or more target antigens exposed on tumor microenvironment cells selected from the group consisting of α-SMA, CD74, CXCL12 and PDGFRβ. Specific.

In a preferred variation of this embodiment, the chimeric antigen receptor (CAR) containing cells were exposed on tumor microenvironment cells and tumor stromal cells selected from the group consisting of α-SMA, CD74, CXCL12 and PDGFRβ. Specific to one or more target antigens.

In a second embodiment, the chimeric antigen receptor (CAR)-comprising cell comprises one or more targets exposed on tumor microenvironment cells selected from the group consisting of CD47, CD51, CD58, CD90, CD206 and CD239. Antigen specific.

In a preferred variation of this embodiment, the chimeric antigen receptor (CAR) containing cells are tumor microenvironment cells and PaCa (pancreatic cancer) cells selected from the group consisting of CD47, CD51, CD58, CD90, CD206 and CD239. Specific to one or more target antigens exposed above. The combination of CD90 and CD239 was found to be particularly specific for PaCa tumor microenvironment cells.

In a third embodiment, the cells containing chimeric antigen receptors (CARs) specific for one or more target antigens exposed on tumor microenvironment cells are further specific for tumor cells with tumor microenvironment cells. be.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows common domains of chimeric antigen receptors (CARs). FIG. 1 shows the general construction concept of chimeric antigen receptors. Schematic diagram showing the function of CAR-T cells with AND logic. FIG. 2 is a schematic diagram showing the function of CAR-T cells with NOT logic. FIG. 13 shows target identification by ultra-high content imaging screening for pancreatic cancer. FIG. 13 shows target identification by ultra-high content imaging screening for pancreatic cancer. FIG. 13 shows target identification by ultra-high content imaging screening for pancreatic cancer.

Definitions Below, the terms "tumor microenvironment cells", "TME cells" and "target cells" are synonymous for cells recognized by (and subsequently attacked by) CAR cells provided by the methods of the present invention. Used.

Such tumor microenvironmental cells (TMEs) grow around or encapsulate solid tumors and contain blood vessels, immune cells, fibroblasts, pericytes, signaling molecules and extracellular matrix (ECM). can be. Scientific definitions can be found in the "NCI Dictionary of Cancer Terms". National Cancer Institute. 2011-02-02 and for a recent review see Am J Cancer Res. Please refer to

Accordingly, the terms "non-tumor microenvironment cells", "non-TME cells" and "non-target cells" are synonymous for healthy cells that are not recognized by (and subsequently attacked by) CAR cells provided by the methods of the present invention. used for

The term "tumor cell" refers to cancer cells that are not part of the "tumor microenvironment cells" and are not, of course, healthy "non-tumor microenvironment cells".

The term "chimeric antigen receptor" or "CAR" refers to modified cells with new specificities derived from sources other than the cell itself.

The term "tumor" is known in medicine as "neoplasm". Not all tumors are cancerous, and benign tumors do not invade adjacent tissues and spread throughout the body.

The term "cancer" refers to a broad group of diseases involving unregulated cell growth. Cancerous cells divide and grow uncontrolled, forming malignant tumors and invading surrounding parts of the body. Cancer can also spread to more distant parts of the body through the lymphatic system or bloodstream.

The term "binding agent" or "specifically binds" or "specific for" with respect to a ligand such as an antibody, fragment thereof, or antigen-binding domain of a CAR recognizes and binds to a particular antigen. refers to an antigen-binding domain that does not substantially recognize or bind other molecules in a sample. An antigen binding domain that specifically binds an antigen from one species may also bind that antigen from another species. This cross-species reactivity does not violate the definition that the antigen binding domain is specific. An antigen binding domain that specifically binds an antigen may also bind different allelic forms of that antigen (allelic variants, splice variants, isoforms, etc.). This cross-reactivity does not violate the definition that the antigen binding domain is specific.

As used herein, the terms "modified cell" and "genetically modified cell" can be used interchangeably. These terms are meant to contain and/or express foreign genes or nucleic acid sequences that modify the genotype or phenotype of a cell or its progeny. In particular, these terms refer to cells, preferably T cells, that have been engineered by recombinant methods known in the art to stably or transiently express peptides or proteins that are not naturally expressed in these cells. Point. For example, T cells are engineered to express an artificial construct, such as a chimeric antigen receptor, on their cell surface. For example, CAR-encoding sequences can be delivered intracellularly using retroviral or lentiviral vectors.

The term "target" as used herein refers to an antigen or epitope associated with a cell that is desired to be specifically recognized by an antigen binding domain, eg, an antibody or the antigen binding domain of a CAR. Antigens or epitopes for antibody recognition may be bound to the cell surface, but may be secreted, part of the extracellular membrane, or shed from the cell.

The term "antibody," as used herein, refers to polyclonal or monoclonal antibodies and fragments thereof, which can be produced by methods known to those skilled in the art. Antibodies can be of any species, eg mouse, rat, sheep, human. For therapeutic purposes, when non-human antigen-binding fragments are used, they can be humanized by any method known in the art. Antibodies may be modified antibodies (eg, oligomers, reduced antibodies, oxidized antibodies and labeled antibodies).

The term "killer cell" as used herein refers to a cell that can kill/destroy/lyse another cell, such as a cancer cell. T cells, NK cells, dendritic cells and macrophages can best be used as killer cells.

The term "modified killer cells" as used herein refers to killer cells that have been genetically modified to be capable of specifically killing target cells, such as tumor cells expressing their respective targets and / or refers to cells modified with CAR to target to kill TME cells.

DETAILED DESCRIPTION OF THE INVENTION The process of the invention allows rapid identification of a set of suitable conjugates and subsequent rapid modification of suitable CAR cells to destroy at least a portion of the TME cells of a cell sample. do. Cell samples comprising TME cells, non-TME cells and non-tumor cells are preferably derived from the same tissue or organ of the same patient.

In order to distinguish TME cells from non-TME cells in a cell sample, a person skilled in the art, such as a pathologist, can determine whether the cell sample contains a tumor and, if so, what part of the cell sample. , can be determined according to known standards or routine methods in oncology. Such criteria include, for example, the guidelines published by the Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften (AWMF) and include, for example, cell morphology, tissue architecture, ploidy, biomarker expression (Her2, p53, BRAC1, APC, EGFR, etc.). obtain.

It is not necessary for the identified conjugates to bind to all TME cells, as tumors can be destroyed even if only a fraction of the TME cells are identified or attacked. Preferably, the process of the invention identifies at least two conjugates with antigen recognition moieties that recognize different antigens that bind to at least 1%, more preferably at least 10%, most preferably at least 30% of TME cells. until it is done.

Although it is desired that the conjugate bind to 0% of non-TME or non-tumor cells, it may be acceptable for the therapy to target some amount of non-tumor/non-TME cells (ie healthy cells). Preferably, the process of the invention binds to TME cells and to at most 10%, more preferably at most 5%, most preferably at most 1% of non-TME cells of the same type of tissue or organ, This is done until at least two conjugates with at least antigen recognition moieties that recognize different antigens are identified. The term "same type of tissue or organ" refers to eg pancreas, liver or stroma.

The process of the present invention has one or more sets of two conjugates with antigen recognition moieties that recognize different antigens, and then at least two identified antigen recognition moieties as chimeric antigen receptors (CARs). Allows rapid identification of one or more different cells. Providing two or more sets of conjugates and two or more CAR cells to increase the likelihood of destroying TME cells and/or down-regulate the antigen to avoid TME cells targeting prevent.

Cells containing a chimeric antigen receptor (CAR) are selected from the group consisting of T cells, NK cells, or cells modified to destroy (lyse) other cells upon being induced to bind to them. can be

In the process of the invention, the cell sample may be subsequently contacted with multiple binding agents in order to characterize the recognition pattern and select binding agents specific for TME cells and binding agents specific for non-TME cells. good. Binding agents so identified can be used for subsequent therapy as defined below.

In a variation of the process, at least two conjugates comprising antigen-recognizing moieties that recognize different antigens bind at least 20% of TME cells and less than 5% of non-TME cells of the cell sample. Preferably, at least two conjugates with antigen recognition moieties that recognize different antigens bind at least 50% of TME cells and less than 5% of non-TME cells of the cell sample. More preferably, at least two conjugates with antigen recognition moieties that recognize different antigens bind at least 70% of TME cells and less than 5% of non-TME cells of the cell sample.

In another variation, at least two conjugates comprise antigen-recognizing moieties that destroy at least 10-fold, preferably at least 50-fold, more preferably at least 100-fold more TME cells than non-TME cells of the cell sample.

The process of the invention involves identifying at least two conjugates with antigen recognition moieties that recognize different antigens that bind to TME cells but do not or substantially do not bind to non-TME cells.

If the CAR comprises at least two antigen recognition moieties, the CAR can be triggered to run at different logic gates. Logic gates have been described in the literature as "AND", "NOT" and "OR" type CAR cells. As will be disclosed in more detail later, in the process of the invention, at least two antigen recognition moieties are provided as AND type, OR type or NOT type and/or at least two antigen recognition moieties are provided as ADAPTER type. . At least two antigen recognition moieties in all these variants are provided as chimeric antigen receptors (CARs).

chimeric antigen receptor (CAR)
As shown schematically in Figures 1 and 2, a chimeric antigen receptor (CAR) can comprise an extracellular domain, including an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular domain can be joined to the transmembrane domain by a linker. The extracellular domain may be linked to a signal peptide.

The term "signal peptide" refers to a peptide sequence that directs the trafficking and localization of a protein within a cell, for example to certain organelles (such as the endoplasmic reticulum) and/or the cell surface.

The term "antigen-binding domain" refers to the region of the CAR that specifically binds to an antigen (thus enabling it to target antigen-bearing cells). A CAR obtained by the present invention may comprise one or more antigen binding domains. Generally, the antigen binding domain on the CAR is extracellular. An antigen binding domain may comprise an antibody or fragment thereof. Antigen binding domains can include, for example, full length heavy chains, Fab fragments, single chain Fv (scFv) fragments, bivalent single chain antibodies or diabodies. Any molecule that specifically binds to a given antigen can be used as the antigen binding domain, such as an affibody, or a ligand binding domain derived from a natural receptor. Often the antigen binding domain is an scFv. Typically, in scFvs, the variable portions of the heavy and light chains of an immunoglobulin are fused by a flexible linker to form the scFv. Such linkers can be, for example, “(G ₄ /S ₁ ) ₃ -linkers”.

The transmembrane domain of the CAR can comprise, for example, sequences of the transmembrane domains of 4-1BB, CD8 and/or CD28. In addition, an activating intracellular signaling domain may be present comprising sequences of the intracellular signaling domain of CD28, CD137 or CD3zeta.

Instead of an activating intracellular signaling domain, the CAR may contain an inhibitory intracellular signaling domain having the sequence of an intracellular signaling domain such as PD1 or CTLA4.

In a preferred variation of this embodiment, the chimeric antigen receptor (CAR) comprises an antigen binding domain specific for CD20 with or without an additional antigen binding domain, wherein the antigen binding domain is a single transmembrane domain and one or more signaling domains. This variant is shown by way of example in FIG. 2A.

In some cases it is beneficial for the antigen binding domain to be derived from the same species in which the CAR is used. For example, if the CAR is intended for therapeutic use in humans, it may be beneficial for the antigen-binding domain of the CAR to comprise a human or humanized antibody or fragment thereof. Human or humanized antibodies or fragments thereof can be produced by various methods known in the art.

The term "spacer" or "hinge" as used herein refers to the hydrophilic region between the antigen binding domain and the transmembrane domain. A CAR identified by the present invention may include an extracellular spacer domain, although it is possible to exclude such a spacer. A spacer can comprise an Fc fragment of an antibody or fragment thereof, a hinge region of an antibody or fragment thereof, a CH2 or CH3 region of an antibody, an accessory protein, an artificial spacer sequence, or a combination thereof. A prominent example of a spacer is the CD8α hinge.

The transmembrane domain of CAR may be derived from any desired natural or synthetic source of such domains. When native in origin, the domains may be derived from any membrane-bound or transmembrane protein.

The cytoplasmic or intracellular signaling domain of the CAR of the invention is involved in the activation of at least one normal effector function of immune cells in which the CAR is expressed. By "effector function" is meant a specialized function of a cell, for example in T cells, the effector function can be cytolytic activity or helper activity, including secretion of cytokines. An intracellular signaling domain refers to the portion of a protein that transmits effector function signals and directs cells expressing the CAR of the invention to perform a specific function. An intracellular signaling domain may include any intact or truncated portion of a given protein's intracellular signaling domain sufficient to transmit an effector function signal.

Prominent examples of intracellular signaling domains used in CARs include the cytoplasmic sequences of T-cell receptors (TCRs) and co-receptors that act in concert to initiate signaling after antigen receptor ligation. .

For CARs with at least two antigen binding domains, the antigen binding domains may have different functions. The process of the present invention shortens the development time of new CAR cells suitable for tumor therapy, which can be provided as "AND" type, "NOT" type, "OR" type CAR cells.

In one embodiment of the invention, the CAR cell is activated, ie the signal of the signal domain is triggered, only when both antigen binding domains bind their respective antigens. Such CAR cells are hereinafter referred to as "AND-CAR". Antigen-binding domains of "AND-CAR" cells are identified in the methods of the invention, e.g., by identifying two antigens on TME cells that are not expressed, or at most one, is expressed in healthy cells. can be done. Figure 3 (published in Transl Cancer Res 2016;5(S1):S61-S65) shows that AND-CAR is inactivated by healthy cells (i) and (ii), but activated by TME cells (iii). is shown.

In another embodiment of the invention, the CAR cells are activated, ie the signaling of the signaling domain is triggered, only when one antigen binding domain binds to its respective antigen and the other does not. Such CAR cells are hereinafter referred to as "NOT-CAR". Antigen-binding domains of "NOT-CAR" cells can be identified in the methods of the invention, for example, by identifying two antigens on healthy cells, only one of which is expressed on TME cells. Figure 4 (published in Transl Cancer Res 2016;5(S1):S61-S65) shows that NOT-CAR is inactivated by healthy cells (i) but activated by TME cells (ii). It is shown.

In yet another embodiment of the invention, CAR cells are already activated, ie the signal of the signal domain is induced, when only one of the two antigen binding domains binds to the respective antigen. Such CAR cells are hereinafter referred to as "OR-CAR". Antigen-binding domains of "OR-CAR" cells can be identified in the methods of the invention, eg, by identifying two antigens that are on TME cells but not on healthy cells.

A further reduction in processing time can be achieved using the so-called "ADAPTER CAR" technique. "ADAPTER CAR" cells contain a signaling domain, a transmembrane domain, a linker domain, and do not have antigen recognition properties. One or more antigen recognition moieties are then conjugated to this linker domain (linker + molecule = adapter). Thus, in the process according to the invention, at least two antigen recognition moieties as chimeric antigen receptors (CAR) can be provided as ADAPTER type.

In a variation of the invention, a cell is provided that contains biotin as the antigen-recognition moiety of a chimeric antigen receptor (CAR), a conjugate of at least two identified antigen-recognition moieties and an anti-biotin moiety is provided, the conjugate is Conjugation of the gate to the cell provides the cell with at least two specified antigen-recognition moieties as a chimeric antigen receptor (CAR).

In another variant of the invention, a cell comprising biotin as the antigen-recognition portion of the first chimeric antigen receptor (CAR) and the second epitope as the antigen-recognition portion of the second chimeric antigen receptor (CAR). at least two identified antigens by providing a conjugate of at least two identified antigen-recognition moieties, an anti-biotin moiety and a second moiety, and conjugating said conjugate with said cell; The recognition moiety is provided on the cell as a chimeric antigen receptor (CAR).

Preferred linkers are molecules that do not exist in the mammalian (human) body or have a low affinity for natural antibodies in the mammalian (human) body. For example, biotin or thiamine units (chemically modified or unmodified) are suitable, identified by suitable adapters such as anti-biotin or anti-thiamine antibodies that are conjugated to desired conjugates with antigen recognition moieties.

The "ADAPTER CAR" technology is disclosed in WO2018078066, which uses the term "linker/labeled epitope" (LLE) for linkers bound to CAR cells.

Preferably, LLE does not provoke an immune response in subjects intended to be treated with cells expressing CAR. This is the extracellular LLE-binding domain of the CAR derived from a natural epitope recognition molecule such that the LLE binds with higher preference than an endogenous labeling moiety without a linker moiety, i.e. a natural molecule (self-antigen) within the subject. can be achieved by using

For example, the extracellular LLE binding domain of CAR binds said LLE with at least 2-fold, preferably at least 5-fold, more preferably at least 10-fold higher affinity than said natural molecule.

For "ADAPTER" CAR cells, CAR cells with linker units and adapters are applied in parallel to the patient. An advantage of this embodiment is that CAR cells with linkers can be generated independently of the identified antigen recognition moieties and need not be developed from scratch. Only adapters need to be generated and tested. This approach also allows control of the intensity and kinetics of therapy, similar to conventional chemotherapy.

Detection Process The methods of the present invention require quenching of the fluorescence emitted from the fluorescent moiety of the cell-bound conjugate. This step allows multiple potential binders or conjugates to be explored until the best combination of conjugates (ie, antigen-binding portions, ie, TME cell antigens) is determined. The term "quenching fluorescence" refers to any method of quenching the fluorescence emission of a cell sample, including removing bound conjugate from the fluorescent moiety, e.g., by enzymatically cleaving the conjugate. Such conjugates are disclosed, for example, in EP-A-3037821, EP-A-16203689.1 or EP-A-16203607.3.

In that variant, a conjugate comprising an affinity system, eg biotin/anti-biotin, can be used, the affinity system being inhibited by the addition of a free competing molecule (such as biotin in the biotin/anti-biotin system). In either case, fluorescence is quenched by releasing the fluorescent moiety from the conjugate and subsequently washing away the "released" fluorescent moiety.

An alternative method of "quenching fluorescence" involves adding an oxidizing agent, such as hydrogen peroxide, or supplying radiation of an appropriate wavelength (such as UV radiation) to quench the fluorescence to the extent that it is incapable of emitting fluorescent radiation. Chemically destroy the part.

Use for treating cancer The cell population obtained by the method of the present invention can be preferably used as a pharmaceutical composition in a method for treating human cancer, particularly human pancreatic cancer or human stromal cancer.

The pharmaceutical composition preferably comprises a population of modified cells expressing CAR obtained by the method of the invention. In a variant of the invention, the pharmaceutical composition is used in the treatment of cancer in combination with chemotherapeutic agents, radiation or immunomodulatory agents.

Recognition of TME by pharmaceutical compositions can trigger responses such as secretion of molecules by CAR cells, which recruit additional cells and reduce the effectiveness of TME in protecting cancer cells. Finally, cancer cells can be attacked by, for example, appropriate CAR cells.

TME cells of cancers to be treated include hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML). , lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other hematologic and solid tumors, or any combination thereof. In another embodiment, the cancer is leukemia (e.g. chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML) or chronic myelogenous leukemia (CML)), lymphoma (e.g. mantle cell lymphoma). , non-Hodgkin's lymphoma or Hodgkin's lymphoma) or hematologic cancers such as multiple myeloma, or any combination thereof. In addition, cancers of the oropharyngeal (tongue, oral cavity, pharynx, head and neck), gastrointestinal (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory cancer (larynx, lung and bronchi), bone and joint cancer, soft tissue cancer, skin cancer (melanoma, basal and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing) sarcoma), tumors of the central nervous system (cerebral neuroblastoma, astrocytoma, glioblastoma, glioma), and breast, reproductive system (cervix, corpus uterus, ovary, vulva, vagina, prostate , testicles, penis, endometrium), urinary system (bladder, kidneys and renal pelvis, ureters), eyes and orbits, endocrine system (thyroid), and brain and other nervous system cancers, or any combination thereof can contain.

Treatment of cancer can include any method that uses at least one of the disclosed antigens or any combination of the disclosed antigens as target molecules. Such methods can be, for example, treatment with agents that bind to an antigen and affect the viability of cancerous cells expressing this antigen, preferably killing the cancerous cells. Examples are the previously disclosed oncolytic viruses, BiTE®, ADCC and immunotoxins.

A subject's immune cells, eg, T cells, can be isolated for treatment. The subject may be afflicted with the above cancers or may be a healthy subject. These cells are genetically engineered in vitro or in vivo to express one or more CARs of the invention. These modified cells can be activated and expanded in vitro or in vivo. In cell therapy, these modified cells can be injected into a recipient in need thereof. These cells may be a pharmaceutical composition (the cells described above plus a pharmaceutically acceptable carrier). The injected cells can kill (or at least arrest the growth of) cancerous cells expressing one or more of the disclosed antigens in the recipient. The recipient can be the same subject from which the cells were obtained (autologous cell therapy) or another subject of the same species (allogeneic cell therapy).

Examples The following examples are intended to provide a more detailed description of the invention, but the invention is not limited to these examples.

Fresh frozen pancreatic cancer specimens were sliced and fixed with acetone. Subsequent screening was performed on the MACSima™ Ultra High Content Imaging Platform (Miltenyi Biotec B.V. & Co. KG) using serial staining of 90 antibodies, 9 of which are shown. Nuclei were stained with DAPI.

Figure 5 shows that a combination of markers CD47, CD51, CD58, CD90, CD206 and CD239 can be used to distinguish TME and tumor cells from healthy cells. In particular, the combination of CD90 and CD239 was specific to tumor microenvironment cells, as both show overlapping expression in stromal cells but not in other cell types. By combining these markers with tumor cell specific markers or combinations of markers, TME and optionally tumors can be specifically attacked.

Claims (15)

A process for providing cells containing chimeric antigen receptors (CARs) specific for one or more target antigens exposed on tumor microenvironment cells, said cell sample comprising tumor microenvironment cells and non-tumor microenvironment cells. prepare the
- contacting the tissue with a conjugate comprising a fluorescent moiety and an antigen recognition moiety,
- removing unbound conjugate from said tissue and detecting cells bound to said conjugate by fluorescence emission emitted from said fluorescent moiety of the first conjugate;
- the step of quenching the fluorescence emitted from said fluorescent moiety of said conjugate comprises antigen recognition moieties that recognize different antigens and in combination can distinguish between tumor microenvironment cells and non-tumor microenvironment cells. , repeatedly until at least two conjugates are identified,
A process characterized by providing a cell having at least two identified antigen-recognizing moieties as a chimeric antigen receptor (CAR). said chimeric antigen receptor (CAR)-containing cells are selected from the group consisting of T cells, NK cells, or cells modified to destroy said other cells upon being induced to bind to them 2. The process of claim 1, characterized by: 3. Process according to claim 1 or 2, characterized in that said at least two antigen recognition moieties are provided as AND-type, OR-type or NOT-type. 4. Process according to any one of claims 1 to 3, characterized in that said at least two antigen recognition moieties are provided as ADAPTER type. providing a cell containing biotin as an antigen-recognition portion of a chimeric antigen receptor (CAR), providing a conjugate of the at least two identified antigen-recognition moieties and an anti-biotin moiety, and combining the conjugate with the cell; 5. Process according to claim 4, characterized in that the conjugation provides the cell with the at least two identified antigen-recognition moieties as a chimeric antigen receptor (CAR). providing a cell containing biotin as the antigen-recognition portion of the first chimeric antigen receptor (CAR) and a second epitope as the antigen-recognition portion of the second chimeric antigen receptor (CAR); providing a conjugate of one antigen-recognition moiety, an anti-biotin moiety and a second moiety, and conjugating said conjugate with said cell to convert said at least two identified antigen-recognition moieties into a chimeric antigen receptor; 5. A process according to claim 4, characterized in that it is provided in said cells as (CAR). 7. The method of claims 1 to 6, characterized in that said at least two conjugates comprising antigen recognition moieties bind at least 20% of said tumor microenvironment cells and less than 5% of said non-tumor microenvironment cells. A process according to any one of claims 1 to 3. 8. Any of claims 1 to 7, characterized in that said at least two conjugates comprising antigen recognition moieties bind at least 10-fold more tumor microenvironment cells than non-tumor microenvironment cells of said cell sample. 10. The process of paragraph 1. 9. Process according to any one of claims 1 to 8, characterized in that the fluorescent radiation emitted from said fluorescent moiety is extinguished by enzymatically degrading said conjugate. wherein the cells containing chimeric antigen receptors (CARs) specific for one or more target antigens exposed on said tumor microenvironment cells are further specific for tumor cells associated with said tumor microenvironment cells. A process according to any one of claims 1 to 9, wherein said chimeric antigen receptor (CAR)-containing cells are specific for one or more target antigens exposed on tumor microenvironment cells selected from the group consisting of α-SMA, CD74, CXCL12 and PDGFRβ. Process according to any one of claims 1 to 10, characterized in that. wherein the chimeric antigen receptor (CAR)-containing cells are specific for one or more target antigens exposed on tumor microenvironment cells and tumor stromal cells selected from the group consisting of α-SMA, CD74, CXCL12 and PDGFRβ. 12. Process according to claim 11, characterized in that it is a target. said chimeric antigen receptor (CAR) containing cells are specific for one or more target antigens exposed on tumor microenvironment cells selected from the group consisting of CD47, CD51, CD58, CD90, CD206 and CD239 Process according to any one of claims 1 to 10, characterized in that wherein said chimeric antigen receptor (CAR)-containing cells are specific for one or more target antigens exposed on tumor microenvironment cells and PaCa cells selected from the group consisting of CD47, CD51, CD58, CD90, CD206 and CD239. 14. Process according to claim 13, characterized in that the preparing a cell sample comprising tumor microenvironment cells, tumor cells and non-tumor microenvironment cells;
- contacting the tissue with a conjugate comprising a fluorescent moiety and an antigen recognition moiety,
- removing unbound conjugate from said tissue and detecting cells bound to said conjugate by fluorescence emission emitted from said fluorescent moiety of the first conjugate;
- quenching the fluorescence emitted from said fluorescent moiety of said conjugate comprising antigen recognition moieties recognizing different antigens, combined to distinguish tumor microenvironment cells and tumor cells from non-tumor microenvironment cells. repeating until at least two conjugates are identified,
11. Process according to claim 10, characterized in that it provides a cell having at least two of the identified antigen recognition moieties as a chimeric antigen receptor (CAR).

Images